Chapter 2: Major histocompatibility complex class I downregulation induced by equine
2.5 Discussion
Over the past 2 decades, a number of viral inhibitors targeting different stages of MHC-I presentation have been characterized (Hansen and Bouvier, 2009). The majority of such inhibitors were found in herpesviruses, although their mechanisms of action vary greatly.
Recently, we discovered that pUL56, encoded by the gene ORF1 of EHV-1, affects MHC-I expression (Ma et al., 2012). Phylogenetic analyses reveal that pUL56 is conserved in many alphaherpesviruses and the pUL56 homologue of EHV-4 also downregulates MHC-I at the cell surface (Said et al., 2012). It was speculated that loss of cell surface MHC-I is induced by endocytosis in the early stages of EHV-1 infection (Rappocciolo et al., 2003), and here, we investigated the putative relationship between pUL56 and endocytosis that might result in the reduction of MHC-I on the cell surface. By using inhibitors that disrupt endosomal or lysosomal function, we initially determined that endosomal acidification and lysosomal proteolysis govern the fate of internalized MHC-I. This finding was further corroborated by confocal microscopy showing that MHC-I co-localized with the lysosome marker LAMP-1.
Endocytosis is an important intracellular transport mechanism that initiates signal transduction and internalization of a number of nutrients, lipids, membrane proteins and pathogens (Doherty and McMahon, 2009). The endocytic pathways that allow infectious entry have been extensively studied for a number of viruses (Mercer et al., 2010); however, it is less well understood how viruses induce endocytosis of key immune-related receptors to achieve immune evasion. Notable exceptions are the KSHV K3 and K5 proteins, which promote MHC-I endocytosis in infected cells. It was shown in transiently transfected cells that these two virus-encoded enzymes directly increase endocytic activity and lead to the uptake of MHC-I molecules from the cell surface (Coscoy and Ganem, 2000). Likewise, the HIV Nef protein was shown to downregulate MHC-I through an endocytic pathway (Blagovesh- chenskaya et al., 2002), which may imply that enhancing endocytosis as a means of preventing or reducing presentation of antigenic (viral) peptides by MHC-I might be evolutionarily conserved among many viruses.
Chapter 2: Major histocompatibility complex class I downregulation induced by equine herpesvirus type 1 pUL56 is through dynamin-dependent endocytosis
We were able to demonstrate here that EHV-1 pUL56 co-localizes from 4 h p.i. with MHC-I-containing vesicles and the lysosomal marker LAMP-1 in infected cells. This co-localization correlates with decreased cell surface MHC-I levels. The results, therefore, suggest that it is indeed pUL56 that mediates the trafficking of MHC-I molecules to lysosomes for degradation. The two known viral MHC-I inhibitors encoded by alphaherpesviruses, ICP47 and pUL49.5, prevent translocation of antigenic peptide into the ER and formation of the tri-molecular complex. Specifically, ICP47 is present in the cytoplasm and exhibits high affinity to TAP, which results in functional inactivation of peptide transport into the ER (Ahn et al., 1996; Tomazin et al., 1996). For pUL49.5, although it also restricts the supply of peptides to the ER, homologues in different viral species are mechanistically diverse. For example, BHV-1 pUL49.5 reduces TAP stability by promoting its proteasomal degradation (Koppers-Lalic et al., 2005), whereas pUL49.5 of EHV-1 and -4 inhibit the recruitment of ATP, which is indispensible for TAP activity (Koppers-Lalic et al., 2008). Unlike ER-based interference with MHC-I maturation, pUL56 is predominantly localized to the Golgi apparatus and endosomal vesicles. It does not perturb peptide transport to the ER (Ma et al., 2012; Said et al., 2012), and its mechanism of action is clearly distinct from those of ICP47 and pUL49.5. Therefore, pUL56 has a novel mode of action with respect to MHC-I downregulation in members of the Alphaherpesvirinae and functions more like gammaherpesviral K3 and K5 by promoting endocytosis.
Based on their constituent elements, endocytic pathways are classified into categories that include clathrin-, caveolae- and Arf6-dependent endocytosis as well as phagocytosis and macropinocytosis (Mayor and Pagano, 2007); however, a growing body of evidence now suggests that unidentified endocytic pathways might exist, and thus the classification scheme has been simplified by discriminating only between clathrin-dependent endocytosis (CDE) and clathrin-independent endocytosis (CIE) (Le Roy and Wrana, 2005). Endocytosis inhibitors are commonly used to investigate endocytic pathways responsible for homeostasis of cell surface proteins, but attention should be given to their possibly pleiotrophic effects (Vercauteren et al., 2010). Given that the turnover of cell surface proteins is dynamically regulated under normal physiological conditions, we tested the influence of our inhibitors on the metabolism of MHC-I, which might distort the interpretation of our results in infected cells. Except for the significant MHC-I reduction seen with the treatment of chloroquine, the other reagents used showed no or very little effect on the presence of MHC-I on the cell surface of mock-infected cells (Fig. 2.S1), ensuring that they target only MHC-I in infected cells. With inhibition of virus-induced MHC-I downregulation by chlorpromazine and Pitstop2 as well by dominant negative molecules, we demonstrated that MHC-I down- regulation is not through the CDE pathway. This observation for EHV-1 differs from that for KSHV, where the K3 protein interferes with the classical CDE pathway (Duncan et al., 2006),
Chapter 2: Major histocompatibility complex class I downregulation induced by equine herpesvirus type 1 pUL56 is through dynamin-dependent endocytosis
while a unique Arf6-dependent pathway is targeted by HIV-1 Nef to divert mature cell surface MHC-I to lysosomes for degradation (Blagoveshchenskaya et al., 2002). Despite the differences, lysosomes are the ultimate cellular compartment where degradation of MHC-I occurs in the case of KSHV, HIV-1, and EHV-1.
Caveolae-dependent endocytosis is considered an important CIE pathway. Along with the involvement of dynamin, caveolin-1 is primarily responsible for the formation of the flask-shaped caveolae (Liu et al., 2002). Moreover, tyrosine kinase activity is necessary for the aggregation and fusion of caveolae or caveolar vesicles (Nomura and Fujimoto, 1999).
Therefore, it is not surprising that a number of viruses gain access to host cells by non-classical endocytic pathways that are all dependent on the action of dynamin and tyrosine kinases but differ in other factors involved (Azab et al., 2013; Damm et al., 2005; Mulherkar et al., 2011; Qie et al., 1999). Here, we studied the role of caveolae-dependent endocytosis in virus-induced MHC-I downregulation. Despite the various effects of four inhibitors for CIE, transfection of a specific dominant negative form of caveolin-1 was unable to rescue cell surface expression of MHC-I, suggesting that caveolae-dependent endocytosis is not involved in MHC-I internalization. In contrast, we found that the decrease of surface MHC-I was remarkably attenuated when the action of dynamin was inhibited with dynasore or a dominant negative form of dynamin. These findings led us to conclude that MHC-I downregulation is associated with dynamin, which facilitates vesicle scission at the plasma membrane and as such is integral to many endocytic pathways (Hinshaw and Schmid, 1995; Marks et al., 2001).
To our knowledge, our report describes the first example of an alphaherpesvirus that downregulates cell surface MHC-I through an endocytic pathway, which is associated with dynamin but unrelated to clathrin and caveolae. However, there might be another unknown viral protein(s) that can drive MHC-I downregulation through endocytic processes, as knockout of pUL56 alone cannot completely prevent endocytosis of MHC-I mediated by EHV-1 infection.
Ubiquitination determines the destiny of endocytosed membrane proteins (Strous and Govers, 1999). On the one hand, the formation of vesicles at different stages of endocytosis requires mono- or poly-ubiquitination (Hicke, 2001). On the other hand, the nature of ubiquitin linkage to lysine results in cargo sorting and degradation either in the proteasome or in the lysosome (Pickart and Fushman, 2004). KSHV K3 and K5 are E3 ubiquitin ligases that directly bind to membrane substrates and trigger endocytosis (Duncan et al., 2006; Means et al., 2007). In the case of HSV-2, pUL56 is able to increase ubiquitination of the E3 ligase Nedd4. This interaction is thought to affect protein sorting or vesicle trafficking but does not affect virus release, as originally surmised (Ushijima et al., 2008). Here, EHV-1 pUL56, which is structurally similar to its HSV-2 orthologue, was shown to direct MHC-I molecules for
Chapter 2: Major histocompatibility complex class I downregulation induced by equine herpesvirus type 1 pUL56 is through dynamin-dependent endocytosis
lysosomal degradation. This process is greatly affected when ubiquitination is chemically blocked. It is therefore tempting to speculate that the function of pUL56 depends on its interaction with an E3 ligase, a hypothesis that we are currently testing.
To address the specificity of pUL56 action, several other cell surface markers were screened in this study. We found that pUL56 can reduce the expression of CD46 and CD63 on the cell surface. CD46 is critical for efficient T cell responses and bridges between the complement system and cellular immunity (Riley-Vargas et al., 2004); for instance, expression of CD46 facilitates destruction of the cells infected with measles virus by CTLs, a process that depends on MHC-I antigen presentation (Cardoso et al., 1996). CD63 is a membrane protein containing four transmembrane domains (tetraspanin), which is widely distributed on endosomal membranes and known to modulate immune signaling pathways (Levy and Shoham, 2005; Pols and Klumperman, 2009). In dendritic cells, for example, intracellular transport of CD63 is associated with antigen presentation by MHC-II (Engering and Pieters, 2001). The implication of pUL56 in downregulating CD46 and CD63 may suggest that it functions as a more promiscuous immune modulator, a notion that is supported by the results obtained in equids infected with the Ab4 mutant unable to express the protein (Soboll Hussey et al., 2011).
Removal of cell surface MHC-I seems a wise strategy for persistent infection, as CTLs would be unable to eliminate infected cells due to a failure to recognize antigens. However, cells devoid of surface MHC-I are unlikely to survive and might be subject to clearance by natural killer (NK) cells. KSHV K5 targets other surface molecules to counteract the threat from NK cells. Along with CD31, CD86, CD144, CD166, and intracellular adhesion molecule 1 (ICAM1) (Means et al., 2007), KSHV K5 also downregulates activation-induced C-type lectin (AICL), MHC-I polypeptide-related sequence A (MICA), and MICB, which are required for NK cell lysis (Nathan and Lehner, 2009; Thomas et al., 2008). Whether pUL56 targets these surface molecules sensed by NK cells remains to be explored.
Taken together, we investigated the pathway by which pUL56 induces downregulation of cell surface MHC-I. The endocytic process responsible for MHC-1 removal from the cell surface is mediated by dynamin but not clathrin or caveolae. Importantly, tyrosine kinase activity and membrane-bound cholesterol are required for MHC-I endocytosis as is ubiquitination. The latter may imply that pUL56 function is dependent on the E3 ligase activity. Since pUL56 is able to regulate other cell surface molecules, it may have a more comprehensive role in regulation of the immune response to infection. Future studies will focus on how pUL56 interacts with ubiquitination and the yet-unidentified viral protein(s) it needs to fulfill its functions.
Chapter 2: Major histocompatibility complex class I downregulation induced by equine herpesvirus type 1 pUL56 is through dynamin-dependent endocytosis
Acknowledgements
We thank Dr. Walid Azab for helpful suggestions on inhibitors and maintenance of equine cells. We express gratitude for the Pitstop2 inhibitors, kindly provided by Dr. Volker Haucke from the Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin-Buch, Germany, and the anti-MHC MAb CZ3, provided by Dr. Douglas F. Antczak (Cornell University, USA). We also gratefully acknowledge the dominant negative mutants kindly provided by Dr. Mark A.
McNiven (Mayo Clinic, Rochester, MN, USA) and Dr. Alexandre Benmerah (Hôpital Necker-Enfants Malades, Paris, France).
T.H. was supported by a grant from the China Scholarship Council. This study was financed by DFG grant OS143/3-1 and unrestricted funds of Freie Universität Berlin to N.O.
Author contributions
T.H., G.M. and K.O. jointly conceived the study and designed the experiments. T.H. and G.M.
performed most of the experiments and interpreted the data. M.J.L. assisted in confocal microscopy. A.S. constructed the virus mutant expressing dsRed and tested some inhibitors.
T.H. and G.M. prepared the manuscript; and K.O. revised it critically. All authors provided comments on the paper and gave their final approval to submission.
Chapter 2: Major histocompatibility complex class I downregulation induced by equine herpesvirus type 1 pUL56 is through dynamin-dependent endocytosis
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Supplemental material
Figure 2.S1 Effects of inhibitors on the turnover of MHC-I in uninfected cells. NBL6 cells were treated with the following inhibitors: (A) 5 μM lactacystin, 150 μM chloroquine and 10 mM NH4Cl.
(B) 2 μM bafilomycin A1, 80 μM Dynasore, 10 μg/ml chlorpromazine, 10 μM Pitstop2, 50 μg/ml genistein, 5 μg/ml filipin, 20 μg/ml nystatin, 20 mM MβCD and 10 μM PYR41. After inhibition for 4 h, cells were suspended and reacted with MAb anti-MHC-I, followed by staining with Alexa Fluor 647-conjugated goat anti-mouse IgG. To detect total levels of MHC-I, cells were fixed in 3.5% PFA and permeablized in PBS supplemented with 0.02% saponin before proceeding to incubation with primary antibody. Expression levels of MHC-I were detected by flow cytometry. Data were obtained from three independent experiments and presented as means ± standard deviation.